Effective strategies for low-toxicity, multiply-administered cancer therapies are uncommonly reported1. Encapsulating doxorubicin into liposomes has increased the total tolerated dose2-4, while cardiac toxicity, mucositis and palmar-plantar erythrodysesthesia restrict the maximum lifetime dose and limit the clinical dosing schedule to 10-12 mg/kg/week at intervals of two to six weeks1, 5, 6. Unexpected synergies between the cardiotoxicities of anthracyclines and growth factors such as anti-ErbB2 antibodies have further increased the need to reduce toxicity7. Given the impact of the dose limitations on efficacy, particles with reduced toxicity would facilitate treatment, particularly in recurrence.
To enhance stability of doxorubicin within the particle, we create a complex between the drug and a transition metal, as has previously been reported for doxorubicin with manganese (II) and irinotecan with copper (II)8-10. Creation of a copper-doxorubicin complex during the loading process is particularly attractive, since the formation of the copper (II)-doxorubicin complex has been associated with oxygen radical-mediated stimulation of DNA strand scission, the stimulation of lipid peroxidation mechanisms and resultant toxicities11-13. Formation of a drug-metal complex during loading changes the morphology of the liposomes and subsequently improves circulation lifetime and the accumulation of liposomes in tumors14, 15. Further, a 1:2 complex of copper and doxorubicin with a stability constant of 1016 forms when a neutral pH is created within liposomes16, 17. Yet, at a low pH, such as the pH encountered within a lysosome or tumor, the stable copper:doxorubicin ratio has been reported to change to 1:1 and the stability of the complex decreases17. Here, we track the liposome shell using positron emission tomography (PET) and the drug using multi-spectral fluorescence in order to assess the pharmacokinetics.
Further, the protective coating of liposomes reduces drug diffusion within the tumor, and the impact of liposomal therapy on clinical efficacy has been modest18. We address the dual issues of toxicity and efficacy by applying our stable particle in an aggressive dosing schedule and incorporating two strategies designed to enhance efficacy: mTOR inhibition to slow proliferation19 and therapeutic ultrasound to enhance accumulation and local diffusion20, 21. The aggressive syngeneic Met-1 model is known to be sensitive to rapamycin (which is an mTOR inhibitor); however, rapamycin alone is not curative in this model22.
Ultrasound, as a source of thermal and mechanical energy can augment drug delivery by releasing the drug or increasing vascular permeability and thus particle accumulation and diffusion20, 21. Tumor blood vessels present relatively permeable capillaries that allow macromolecules and small liposomes (100 nm) to leak through open gaps and fenestration due to the enhanced permeability and retention (EPR) effect23, 24. Heating of the tumor rim, when combined with liposomal drugs, can enhance therapeutic efficacy as was previously demonstrated for radiofrequency (RF) ablation combined with liposomal doxorubicin25. Thus, by enhancing the pharmacokinetic profile and the extent of the EPR effect, we demonstrate enhanced efficacy and reduced toxicity in a highly aggressive mouse model of breast cancer26, 27.